revealing the diversity of amber source plants from the early … · 2020. 8. 20. · research...

Seyfullah et al. BMC Evolutionary Biology (2020) 20:107 https://doi.org/10.1186/s12862-020-01651-2

RESEARCH ARTICLE Open Access

Revealing the diversity of amber source

plants from the Early Cretaceous CratoFormation, Brazil

Leyla J. Seyfullah1* , Emily A. Roberts1,2 , Alexander R. Schmidt3 , Eugenio Ragazzi4 , Ken B. Anderson5,Daniel Rodrigues do Nascimento Jr.6, Wellington Ferreira da Silva Filho6 and Lutz Kunzmann7

Abstract

Background: Amber has been reported from the Early Cretaceous Crato Formation, as isolated clasts or withinplant tissues. Undescribed cones of uncertain gymnosperm affinity have also been recovered with amber preservedin situ. Here, we provide multiple lines of evidence to determine the botanical affinity of this enigmatic,conspicuous cone type, and to better understand the diversity of amber-source plants present in the CratoFormation and beyond.

Results: A new taxon of amber-bearing pollen cone Araripestrobus resinosus gen. nov. et sp. nov. is described herefrom complete cones and characteristic disarticulated portions. The best-preserved cone portion has both in situamber infilling the resin canals inside the preserved microsporophyll tissues and pollen of the Eucommiidites-type.This places this genus within the Erdtmanithecales, an incompletely known gymnosperm group from the Mesozoic.FTIR analysis of the in situ amber indicates a potential araucariacean conifer affinity, although affinity withcupressacean conifers cannot be definitely ruled out. Pyr-GC-MS analysis of the Araripestrobus resinosus gen. nov. etsp. nov. in situ fossil resin shows that it is a mature class Ib amber, thought to indicate affinities with araucariaceanand cupressacean, but not pinaceous, conifers. This is the first confirmed occurrence of this class of amber in theCrato Formation flora and in South America, except for an archaeological sample from Laguna Guatavita, Colombia.

Conclusions: The combined results of the cones’ novel gross morphology and the analyses of the in situ amberand pollen clearly indicate that the new taxon of resinous gymnosperm pollen cones from the Crato Formation isaffiliated with Erdtmanithecales. The cone morphology is very distinct from all known pollen cone types of thisextinct plant group. We therefore assume that the plant group that produced Eucommiidites-type pollen is muchmore diverse in habits than previously thought. Moreover, the diversity of potential amber source plants from theCrato Formation is now expanded beyond the Araucariaceae and the Cheirolepidiaceae to include this member ofthe Erdtmanithecales. Despite dispersed Eucommiidites pollen being noted from the Crato Formation, this is the firsttime macrofossils of Erdtmanithecales have been recognized from the Early Cretaceous of South America.

Keywords: Amber, Araucariaceae, Cheirolepidaceae, Erdtmanithecales, Eucommiidites, Gnetales, In situ pollen, Resin

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

* Correspondence: [email protected] of Palaeontology, University of Vienna, Althanstraße 14, 1090Vienna, AustriaFull list of author information is available at the end of the article

http://crossmark.crossref.org/dialog/?doi=10.1186/s12862-020-01651-2&domain=pdfhttp://orcid.org/0000-0002-0199-9923https://orcid.org/0000-0003-0604-8331https://orcid.org/0000-0001-5426-4667https://orcid.org/0000-0002-0390-6823https://orcid.org/0000-0001-6445-3920http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/publicdomain/zero/1.0/mailto:[email protected]

Seyfullah et al. BMC Evolutionary Biology (2020) 20:107 Page 2 of 22

BackgroundAmber (fossilized plant resin) has rarely been reported fromSouth America. The only significant fossiliferous depositthat has been described is from the middle Miocene PebasFormation of Peru and is thought to be angiosperm inorigin [1]. This amber contains inclusions of diverse arthro-pods, pollen and spores [1]. Small amounts of amber arerecorded from the Miocene of the Pirabas Formation nearCapanema, Pará, Brazil, and are thought to derive fromthe angiosperm Hymenaea (Leguminosae) [2, 3]. Oligo-Miocene amber from Pecket Mine (Punta Arenas), Chile,may have affinities to podocarpalean conifers [4]. Amberis also present inside fossil conifer (Agathis) tissues recov-ered from the early Eocene Laguna del Hunco flora andthe early middle Eocene Río Pichileufú flora, both innorthwestern Patagonia, Argentina [5].There are also undated fossil resins reported from South

America. These may be ambers or sub-fossil resins(copals), or even resins; all are angiosperm-derived. TheGuayaquil (Ecuador) deposit is thought to have affinities toProtium (Burseraceae) and the two Colombian deposits(Gíron and Medellín) are both thought to be derived fromHymenaea [2].Older Early Cretaceous (upper Aptian-lower Albian)

South American amber has been reported from Brazil([6] and references therein). There are three EarlyCretaceous ambers from Brazilian locations: Amazonas(Alter do Chão Formation), Araripe (Crato Formation) andRecôncavo (Maracangalha Formation, Caruaçu Member).Using GC-MS, affinities with conifers were suggested for allthree ambers including Araucariaceae, Cupressaceae orCheirolepidiaceae, although in case of the Recôncavoamber, a potential Podocarpaceae origin was additionallyinferred [7].Here, we focus on amber-bearing cone fossils from the

Early Cretaceous of the Araripe Basin, Ceará, Brazil.Amber occurs as isolated clasts and preserved withinplant fossils [8]. This amber was thought to be derivedfrom araucariacean plants, as both the chemical analysisof isolated amber clasts and the associated macrofossilswith in situ (but chemically untested fossil resin) appearto support this [8], although [7] broadened the potentialaffinities of the amber source plants. The presence ofresin canals within the tissues of the conifers Pseudofre-nelopsis (Cheirolepidiaceae) and Brachyphyllum obesum(Araucariaceae) present in the Crato Formation flora hasalso been shown [9]. This suggests that both theseconifer families could potentially be sources for theCrato Formation amber clasts.We investigated previously undescribed cones with

amber preserved to understand their biological affinity,and the diversity of potential amber source plantspresent in the Early Cretaceous Crato Formation flora.Using a combination of extracting in situ pollen and

amber analyses, we uncovered the presence of a further,previously unknown potential source plant of amber inthe Crato Formation. The recovered in situ pollen ofEucommiidites-type places the new taxon withinErdtmanithecales.Our study thus contributes to a better understanding of

the habit and bauplan of the extinct Erdtmanithecales andis likely able to add characters to enlighten its relationshipsto Gnetales and Bennettitales (BEG (Bennettitales-Erdtma-nithecales-Gnetales) group, e.g., [10]) as well as to conifers.Of all the gymnosperm groups possibly implicated in theorigin of the angiosperms, the Erdtmanithecales are theleast well-known. They are thought, based on the sharedpresence of chlamydospermous seeds, to possibly have aclose relationship with the Bennettitales and Gnetales [10],although this hypothesis remains controversial.

MethodsEight cones and cone fragments, one of them with bothin situ amber pieces and in situ pollen preserved fromthe C6 limestone unit of the Crato Formation, SantanaGroup, northeastern Brazil were investigated. Both thein situ amber and pollen were extracted and analysed tounderstand the biological affinity of this plant as it hadnot been previously described.The specimens are from the following repositories: The

Museum für Naturkunde Berlin (specimens prefixed with aMB.Pb. number), University of Portsmouth Collection(specimens prefixed with UOP-PAL-MC), SenckenbergResearch Institute and Natural History Museum Frankfurt/M. (specimens prefixed with SM.B), Universidade Estadode Rio de Janeiro (specimen prefixed with UERJ) andFundação Paleontológica Phoenix, Ceará, Brazil (specimensprefixed with FPH). Complete cone specimens investigated:SM.B 16594; MB.Pb.1997/1350; MB.Pb.2020/0020 (rubbercast). Cone fragment specimens investigated: FPH-83-B;MB.Pb.1997/1246; MB.Pb.1999/1440; SM.B 16599; UOP-PAL-MC0004. Additionally, a resinous specimen of Brachy-phyllum obesum (UERJ 15-P1) was used to provide in situamber for the comparison of amber chemistries.

Geological setting and preservationThe Late Jurassic to Early Cretaceous Araripe Basin,northeastern Brazil is a tectonic depression related to theopening of the South Atlantic (e.g., [11]). Its sedimentaryhistory is linked to the rifting processes and can bebroadly subdivided into pre-rift, rift and post-rift tectonicsequences [12]. Recent lithostratigraphic concepts placethe post-rift-phase sediments of the Barbalha, Crato, Ipubiand Romualdo Formations into the late Aptian-earlyAlbian Santana Group [13]. The Crato Formation repre-sents mainly lacustrine carbonates and siliciclastic sedi-ments, which are interfingered, at least at its top, bypossible fan delta deposits [14]. Six informal limestone


units (C1-C6) are distinguished [15], of which C6 at thetop of the formation is commercially important and isexcavated as “Pedra Cariri” in several quarries between thetowns Nova Olinda and Santana do Cariri, Ceará [16].Fossil biota are therefore almost exclusively recorded fromthe C6 limestone, which is the case with our material. Theexact stratum or strata within the C6 is rarely knownbecause most C6-derived fossils are found by workers inthe quarries, who do not exactly record the location of thespecimens. This massively hampers floristic and faunalreconstructions of the Crato ecosystem to date. The entityof lacustrine limestone of the Crato Formation has alsobeen referred to as the fossiliferous Nova Olinda Memberin other literature (e.g., [17]) and has been sometimessubdivided into two units [18].The Crato Formation yields an important Gondwanan

fossil Konservat Lagerstätte [17], and is particularlyconsidered to be late Aptian to earliest Albian (~ 115million years old) in age, based on palynological data[18, 19]. This Konservat Lagerstätte is a para-autochthonousto allochthonous assemblage with diverse insects, verte-brates including pterosaurs, and plants. As mentionedabove, the Lagerstätte is exclusively described to date fromthe C6 limestone, a finely laminated rhythmic carbonatedeposited in shallow water under mainly hypersaline condi-tions [17, 20]. Whether the Crato Lake was exclusively afreshwater lake, or temporarily a brackish lagoon from firstmarine ingressions, was a matter of debate [17, 21] but fullymarine taxa are not present. The assemblage constitutesfossil remains of both aquatic and terrestrial biota, with theterrestrial organisms likely either blown in or drifted in byflood events (e.g. [22]) and ephemeral rivers [15, 23].The plant fossils from the C6 limestone are mostly

preserved as iron monosulfide (usually greigite) re-placements in the unweathered material, or as goethite(hydrated iron oxide) replacements in weathered ma-terial [24]. Coalified (compression) fossils are mainlyrecorded from the basal “Seven-Cuts”, an informallevel of the C6 limestone (pers. comm. from quarryworkers to Wellington Ferreira da Silva Filho). Basedon the sediment characteristics we infer that at leastsome study specimens (FPH-83-B; SM.B 16599) origin-ate from the “Branco” Zone (white zone) of the C6limestone, which is three to four meters above the baseof the C6 limestone. The informal zonation of C6 andits terminology are used by the limestone quarryworkers in order to localize the excavation positionand to distinguish the specific lithological characterand quality of the cut stone. Among the 40 taxa of fossilplants, which are validly published to date from the C6limestone, a pteridophyte [25], cycads [26], diverse coni-fers [27–29], gnetaleans [22, 30–33] and angiosperms[34–37] have been described, along with isolated pieces ofamber [6–8] and charcoalified wood [37].

PhotographySpecimens were photographed with a Canon 60Dcamera. Light microscopy images were taken using aCarl Zeiss Discovery V8 stereo microscope with a Canon80D camera attached.

Collection of amber samplesAmber samples were removed from the cone andBrachyphyllum specimen using sharpened needles, thenscreened and cleaned under a microscope, then sub-jected to chemical analyses. The two chemical analyses(Fourier-transform infrared (FTIR) and Pyrolysis gaschromatography mass spectroscopy (Pyr-GC-MS)) arecommon techniques used to chemically profile the fossilresin to give an indication of the botanical affinity.

Solid-state Fourier-transform infrared (FTIR) analysis ofamberSolid-state Fourier-transform infrared analysis wasperformed on freshly powdered samples of amber froma cone portion included in potassium bromide pellets. APerkin Elmer 1600 Series FTIR Spectrophotometer wasused in the wavelength range 2.5–15 μm (4000–670 cm− 1).

Pyrolysis gas chromatography mass spectroscopy (pyr-GC-MS) of amberPyr-GC-MS analyses were completed using a CDS 2500Pyrolyzer coupled to an Agilent 5890/5973 GC-MS. Pyr-olysis was carried out at 480 °C for 10 s in the presenceof excess tetramethyl ammonium hydroxide (TMAH).Pyrolysis products were separated using a 60m ZB-1701capillary GC column using He as carrier gas in constantflow (1 ml/min) mode and temperature programmed asfollows: 40 °C (4 min), 4 °C/min, 280 °C (16 min). Individ-ual analytes were identified by comparison of MS andretention time data with data for known analytes.

Pollen sampling and preparationSamples of the dark remains of the cone fragment wereremoved using a sharpened needle and prepared forboth scanning electron and light microscopy. Forscanning electron microscopy (SEM), small pieces of thetissue were transferred to a carbon-covered SEM mountusing a wet hair from a superfine brush. The stubs weresputtered with platinum–palladium (2 × 120 s at 20 mA,10 nm coat thickness) using an automatic sputter coater(Canemco Inc.) and examined under a field emissionscanning electron microscope (Carl Zeiss LEO 1530Gemini).For light microscopy (LM), the samples picked with

needles were treated with Schultze solution (concen-trated nitric acid saturated with a few potassium chloratecrystals added) and washed in distilled water, thentreated with 4% ammonia and washed until neutral. The


extracted material was then mounted on microscopyslides in glycerine jelly, and covered with a coverslip.These pollen preparations were observed with a trans-mitted light microscope (Carl Zeiss Axioscope A1)equipped with a Canon 450D digital camera, and aredeposited with the source specimen (MB.Pb.1997/1246).

Individual pollen grain preparationIn order to accurately describe the in situ pollen and itsrange of micromorphology, individual pollen grain prepar-ation was done. Grains were removed from one LM slide(concentrated HCl was used to dissolve the glycerine jellymounting medium) and the grains picked off. Theseselected grains were acetolysed (9 parts acetic anhydride,1 part concentrated sulfuric acid, heated to 100 °C for 4mins) in a drop on a glass slide, then washed and trans-ferred to a glycerine drop for mounting using a dissectingneedle with a nasal hair affixed [38, 39]. After LM photog-raphy, the individual grains were mounted on an SEMstub with a drop of absolute ethanol to remove all tracesof glycerine. The stub was sputtered with gold. Thesegrains were then observed using a FEI Inspect S50 SEMand then remounted in a different orientation, resputteredwith gold and observed again.

ResultsWe describe a new fossil-genus and species, based onpollen cones with both in situ amber and pollen present,from the Crato Formation, and infer their affinity aswithin the extinct order Erdtmanithecales. We also showthat the amber can be classified as belonging to the mostcommon type in the geosphere using pyr-GC-MS,highlighting that there were more source plants for thisamber type than previously recognized.

Systematic paleontologySPERMATOPHYTA.ERDTMANITHECALES E.M.Friis et K.M.Pedersen,

1996.Family unknown.

RemarksThe order Erdtmanithecales was created to accommodatethe family Erdtmanithecaceae E.M.Friis et K.R.Pedersen1996. This family is based on isolated reproductive organsincluding dispersed pollen of the Eucommiidites-type, mi-crosporangiate organs with in situ Eucommiidites-typepollen and chlamydospermous seeds with Eucommiidites-type pollen in the micropyle of some seed specimens. Thisnewly described microsporangiate taxon has in situEucommiidites-type pollen, placing it within the orderErdtmanithecales. However, the new taxon described heredoes not fit well into the family Erdtmanithecaceae since

this has been diagnosed with the ‘male reproductive organsof closely spaced microsporangiate units; microsporangiateunits stalked and peltate, bearing numerous sporangia in aradial arrangement around stalk; dehiscence of sporangiaby longitudinal slits’ [40]. The new taxon has quite a differ-ent arrangement. We place the new taxon within theorder, but choose neither to amend the Erdtmanithecaceaenor to create a separate family at this time. We highlightthe enigmatic morphology of the cones being markedlydistinct from all previously recognized microsporangiateorgans producing Eucommiidites-type pollen. Much morediversity among Erdtmanithecales is also inferred from thediversity of conspicuously differently sized isolated seedsknown from various Early Cretaceous sites [10].

Araripestrobus Seyfullah, E.A.Roberts, A.R.Schmidt etL.Kunzmann gen. nov.

Etymology: From the Araripe Basin, where the CratoFormation flora originates.

Generic diagnosis: Large (up to 170mm long and 74mmwide) microsporangiate pedunculate cones, appearing‘segmented’ or organized into distinct layers in lateral outersurface view, built up of about 10 disc-shaped cone por-tions and a basal and apical cone portion with dome-shaped apex and base respectively, gaps present betweenthe layered portions. Peduncle robust, short, diameter about1/3 of the cone diameter. Detached microsporophylls leaverhomboidal scars with a central impression of a possiblevascular bundle. Individual cone portions found detachedmore or less trapezoid to diamond-shaped in cross-sectionwith a longer and a shorter diameter and the cone axis sub-centrally; outer margins frequently arc-shaped in cross-sectional view. Slender, wedge-shaped microsporophylls, indense facetted arrangement, generally in helical phyllotaxis;particularly each cone portion with 2–4 levels of microspo-rophylls, in alternating positions in adjacent levels. Each mi-crosporophyll with 10 or more longitudinally running resincanals. In situ pollen of Eucommiidites-type, boat-shaped,‘trisulcate’, surface finely perforate, smooth to microrugu-late; microfossulae between perforations.

Type: Araripestrobus resinosus gen. nov. et sp. nov.

Araripestrobus resinosus Seyfullah, E.A.Roberts,A.R.Schmidt et L.Kunzmann gen. nov. et sp. nov.

Etymology: The specific epithet highlights the resinousnature of the cone.

Holotype: specimen MB.Pb.1997/1246, illustrated in Fig. 3a.

Paratype 1: specimen SM. B 16594, illustrated in Fig. 2a.

Paratype 2: specimen MB.Pb.1997/1350, illustrated inFig. 2b-e.

Fig. 1 Reconstruction of Araripestrobus resinosus gen. nov et sp. nov.with terminology used here: L – level of microsporophylls(horizontally aligned microsporophylls, not congruent withphyllotaxis); CP – cone portion (if isolated – detached cone portion);M – microsporophyll; S – scar of detached microsporophyll on coneaxis (dot – putative vascular bundle); P – peduncle; scale bar: 50 mm


Type horizon and locality: Lower Cretaceous (upperAptian-lowermost Albian), C6 limestone horizon,Crato Formation, Santana Group, Araripe Basin,northeast Brazil.

Specific diagnosis: as for genus.

Remark to the selection of types: The most significantspecimen displaying almost all diagnostic characters isselected as the holotype. But, it is only a disarticulatedcone fragment, herein called a detached cone portion(for terminology used see Fig. 1), lacking importantgross-morphological features. Therefore, an almostcomplete cone is additionally selected as the paratype 1and an incomplete cone, a key specimen illustrating theinitial disintegration of cones into distinct portions aswell as resin canals of microsporophylls is selected asparatype 2.Additional material: Detached cone portions: FPH-83-

B; MB.Pb.1999/1440; SM.B 16599; UOP-PAL-MC0004.Complete cone: MB.Pb. 2020/0020 (rubber cast).

DescriptionWe assign eight specimens to the new genus and species,and provide a reconstruction to aid understanding of ourterminology and interpretation due to the unusual morph-ology of the cones and microsporophylls (Fig. 1). Thereare three almost complete cones (Fig. 2) and five disc-likestructures that we term ‘detached cone portions’, whichare fragmentary remains of cones where a portion fromthe cone has become detached, is now observed in trans-verse section, and is composed of several layers of micro-sporophylls that are usually preserved around a cone axis.One detached cone portion (holotype, Fig. 3a) is from un-weathered material and so has both amber and pollenpresent in situ. The four other detached cone portionsshare a similar gross morphology and they are preservedin the same orientation with the central axis perpendicularto the bedding surface, giving a compressed transverseview of the detached cone portions. All the detached coneportions are at least partially three-dimensionally pre-served, with varying amounts of tissue remaining (Figs. 4and 5). Due to the partial nature of these cone remains itis not possible to determine the original shape and any ab-axial or adaxial surfaces in these specimens. Only theholotype has yielded in situ pollen (Figs. 6 and 7). The al-most complete cones, one of which is the paratype 1, rep-resent flattened organs in longitudinal view. Paratype 1 isalmost an impression with nearly no coalified tissueremaining (Fig. 2a); the second cone specimen, paratype 2(Fig. 2b-e), preserved as goethite replacement, nicely ex-hibits the outer cone surface and the beginning ofdisarticulation into the characteristic portions (Fig. 2b ar-rowheads mark areas of separation between the portions);

Fig. 2 Longitudinally preserved cones of Araripestrobus resinosus gen. nov et sp. nov. from the Crato Formation. a Paratype 1 (SM.B 16495),almost complete cone in lateral outer-surface-view, showing sigmoidal-shaped gaps between groups of layered microsporophylls in the lowerpart of the cone (arrowheads). b–e Paratype 2 (MB.Pb.1997/1350). b A median-apical cone part with seven cone portions with a gap betweenthem (white arrowheads), and a rounded dome-shaped apex with a bleb of amber towards the right side of the specimen apex (blackarrowhead). c Median cone part displaying individual cone portions with two to four horizontal rows of microsporophylls and a gap betweenthese portions. d Polygonal-hexagonal outline of cross-sections of microsporophylls. e Upper cone part showing microsporophylls with exposedresin canals. f A rubber cast (MB.PB.2020/0020) of a basal-median part of a cone including a peduncle, arrowheads indicate gaps developingbetween microsporophyll layers


Fig. 3 Holotype of Araripestrobus resinosus gen. nov et sp. nov. (MB.Pb.1997/1246) from the Crato Formation with in situ pollen and amberpresent. a Apically compressed detached cone portion with central axis (dark central oval) and the remains of microsporophylls (arrowheads).b Variously coloured amber pieces embedded within the darker brown plant tissue remains at the margin of the portion, amber clearly in rows(arrowheads) indicating the position of resin canals. c Resin canals infilled with fossil resin within the plant tissue remains showing that thedensely packed canals run from the microsporophyll margin towards their interior (3 stacked images). d–f Microsporophylls with amber andvarious outlines (e 9 stacked images, f 5 stacked images)


and the third specimen is a rubber cast from a cone fromwhich microsporophylls are partly detached displayingthe cone axis and the phyllotaxis of microsporophylls(Fig. 2f). Based on the identical microsporophyllmorphology of the detached cone portions and the al-most complete cones, all fossils belong to the sametaxon.

Complete conesThese three laterally compressed specimens are more orless cylindrical in outline, but none are complete. Giventhe unusual morphology of the cones we provide a dia-grammatic reconstruction (Fig. 1) based on a compositeof these specimens to aid with terminology and inter-pretation. Paratype 1 (SM.B 16495, Fig. 2a) is an almost

Fig. 4 Araripestrobus resinosus gen. nov et sp. nov. from weathered material (UOP-PAL-MC0004) a Apically compressed detached cone portionwith central axis (dark central oval) and the remains of microsporophylls. b Microsporophyll remains radiating from the central axis (upper leftside), arrowheads indicating fibrous nature of microsporophylls. c Better preserved microsporophylls at the edge of the detached cone portionshowing more detail of the microsporophyll shape and structure. d Detail of a thick microsporophyll with now empty resin canals seen inoblique view as oval voids (arrowheads). e Scanning electron micrograph of a microsporophyll showing resin (arrowhead) inside a canal


Fig. 5 Other detached portions of cones assigned to Araripestrobusresinosus gen. nov et sp. nov. a Specimen SM.B. 16599 is an almostcompletely preserved rather diamond-shaped detached coneportion. b Specimen FPH-83-B is an ovoid-diamond-shapeddetached cone portion in cross-sectional view. c A fragment of adetached cone portion, MB.Pb.1999/1440


complete cone in lateral outer-surface-view 170mm longand up to 66 mm wide. Its shape is cylindrical to slightlyovoid with widest width after approximately one-third ofthe cone length. The apex and base are not completelypreserved but from a geometrical point of view, andcompared to the other specimens, only small parts aremissing. It is composed of 12 different layers of groupedmicrosporophylls that we equate with the smaller dis-persed cone portions. The horizontal outline of some ofthe median portions is somewhat sigmoidal but the

outline of the apical portions is rather horizontal in rela-tion to the long cone axis (Fig. 2a). Individual portionsare 10–12mm high, only the uppermost portion mea-sures 16 mm in height and is not complete. Each portioncontains at least two adjacent levels of microsporophyllswith their shapes corresponding to the shape of thosefrom isolated cone portions described above. We call ahorizontal set of microsporophylls a ‘level’ (Fig. 1) in-stead of a ‘row’ because the latter term could be mislead-ing implying a whorled phyllotaxis of microsporophylls.Individual portions of the cone are markedly separatedby a narrow gap, giving the segmented or layered ap-pearance (Fig. 2a). The overall shape and arrangement ofmicrosporophylls at the cone appears facetted. The out-line of the distal ends (heads) of microsporophylls is al-most hexagonal isodiametric but appears somewhatirregular most likely due to taphonomic deformation ofthe cones, the surface is almost smooth. The phyllotaxisof the microsporophylls within a cone segment is notclearly observable; however, the alternating arrangementof microsporophylls of adjacent levels excludes arrange-ment in vertical rows (orthostichies).The apical part of the cone is best seen in Paratype 2

(MB.Pb.1997/1350, Fig. 2b), measuring 105 mm long and63mm wide, with seven cone portions present. The ap-ical cone portion shows a rounded dome-shaped apexand consists of more horizontal microsporophyll levelsthan the other portions, about 5–6. While the apicalcone portion is 19 mm high, the remaining portions are10.5–15 mm high. The median cone part shows distinctwide gaps at the cone margin, that narrow towards thecone axis, between the individual cone portions. Medialcone portions are three to four microsporophyll levelsthick (Fig. 2c). The median cone portion also displaysthe facetted arrangement of microsporophylls, which arepolygonal-hexagonal in cross-section (Fig. 2d). Micro-sporophylls of the upper cone portions show numerousresin canals per microsporophyll; the arrangement ofwhich is likely in a circle around the microsporophyll’scenter (Fig. 2e). A bleb of amber is seen towards theright side of the specimen apex in contact with a coneportion fragment which is obviously disarticulated fromthis or from another cone. Therefore, it is not clearwhether the amber originated from this specimen.The basal part of a cone including a peduncle is best ob-

served in the third specimen, a rubber cast (MB.PB. 2020/0020, Fig. 2f), measuring 130mm in length and 74mm inwidth. The peduncle part is preserved displaying nofeatures of previously attached leaves. The massive ped-uncle is about 20mm in diameter but could be somewhatflattened and about 42mm long. Eight cone portions arepreserved, the basal most being somewhat higher than theremaining (10.5–15mm). Individual cone portions display2–4 levels of microsporophylls, in alternating

Fig. 6 Araripestrobus resinosus gen. nov et sp. nov in situ pollen. a–d Scanning electron micrographs, e–f light micrographs. a–b Pollen grainsembedded in the cone tissue, arrowheads indicate areas of preserved amber. c Pollen grains in different orientations showing typical main sulcuswith rounded ends (white arrowheads) and lateral pseudosulci with sharp ends (black arrowheads). d Amber-embedded pollen grain showing alateral sulcus and the finely scabrate, punctate ornamentation; e–f Pollen grains adhered to microsporangial surface showing the sulci withrounded ends (arrowhead)


Fig. 7 Individually prepared pollen grains from Araripestrobus resinosus gen. nov et sp. nov. under SEM. a–c Pollen with view of the sulcus.d Detail of c showing smooth perforate sulcus membrane and rounded end. e Pollen grain folded along sulcus showing one pseudosulcus andthe perforate microrugulate surface. f Detail of pseudosulcus area near rounder end from grain in e. g–h Pollen orientated on the sulcus with thetwo pseudosulci visible and variable surface texture. i Detail of h showing the microrugulate surface with both microfossulae and perforations


arrangements between adjacent levels. Considering thecone dimensions, the microsporophylls are approximately15–20mm long. Some microsporophylls from the basal-most cone portions are detached showing the “naked”cone axis with densely arranged rhomboid scars in helicalphyllotaxis. The flattened preservation of the cone doesnot allow us to determine parastichy numbers.

Detached cone portionsThe detached cone portions show cross-sectional in-ternal cone morphology. Their general outline variesfrom ovoid-diamond-shaped (Figs. 3a, 5b), to roughlytriangular (Fig. 4a), to almost oval (Fig. 5a). The outermargins appear arc-shaped (Fig. 5a, c) and roof-shaped(Figs. 3a, 4a); both shapes can be present in one speci-men (e.g. holotype, MB.Pb.1997/1246; Fig. 3a). Addition-ally, the holotype exhibits a small indentation on oneside (Fig. 3a). Length and width of the detached portions

are always dissimilar (see Table 1) measuring 45–65mmby 21–42mm. The central to sub-central axis is roundedto oval in cross-sectional outline (for measurements seeTable 1). The central axis appears to be slightly sunkenfrom the plane of the microsporophylls and slightlycontracted away from them.Tightly packed microsporophylls radiate out from the

axis forming up to four overlapping levels. The distallevel slightly slopes up to the sediment surface. Phyllo-taxis cannot be deduced from the detached cone por-tions; at least the microsporophylls of adjacent levelsseem to be in alternating positions. Microsporophyllsare slender wedge-shaped; the proximal part is not wid-ened like an apophysis but the outer margin is veryslightly curved. If the outer surface is somewhat abradedthe margin appears irregular and rugged/fissured. Thesedistal parts are up to 4.5 mm wide. The likely mostcomplete microsporophyll is 13.5 mm long in

Table 1 Dimensions and preservation state of detached cone portions of Araripestrobus resinosus gen. nov. et sp. nov.

CharacterSpecimen(figure reference)

Preservation Outline / shape ofcone portion

Length [mm] Width [mm] Size of axis incross-section [mm]

MB.Pb.1997/1246(holotype; Fig. 3a)

compression: cuticle, insitu pollen, in situ resin

ovoid-diamond-shaped 57.4 34.6 7.5 × 13.2

UOP-PAL-MC0004(Fig. 4)

oxidizedno in situ resin

almost triangular 54 42 6 × 12

SM.B. 16,599(Fig. 5a)


almost diamond-shaped 61 29.5 6.5 × 6.5

FPH-83-B(Fig. 5b)


ovoid-diamond-shaped 65 32.5 8 × 8

MB.Pb.1999/1440(Fig. 5c)

compression: in situ resin,no cuticle, no in situ pollen

half portion: arc-shaped 45.5 21 (not preserved)


longitudinal lateral view. Each microsporophyll containsseveral longitudinally running resin canals (average 0.1–0.2 mm wide in longitudinal section) filled with a yellow-orange-to red-brown resin. Resin ducts situated withinthe original plant tissues (in situ amber) are apparent inthe holotype (Figs. 3b-e) and in specimen MB.Pb.1999/1440. The number of resin canals is difficult to countbut at least more than 10 canals per microsporophyll arepresent. The ovals of empty channels, seen in specimenUOP-PAL-MC0004 (Fig. 4d), measure 0.1–0.2 mm inwidth and 0.1–0.3 mm length.

In situ pollen grainsDespite pollen sacs not being recovered, in situ pollengrains (Fig. 6) were found clustered within the plant tis-sue (Fig. 6a), alongside pieces of in situ amber (Fig. 6b,arrowheads). The pollen is ovoid to elliptic in outline inequatorial view (22–35 μm long, 10–20.5 μm wide), witha broad sulcus that is often almost closed over by therest of the surface and has rounded ends (Fig. 6c whitearrowheads). In some orientations, the pollen grains arequite folded and even almost appear to have furtherfurrows (Fig. 6c, black arrowheads), but these are notalways present. Under SEM, the pollen often has a per-forate surface becoming finer to almost smooth towardsthe sulcus (Fig. 6c-d). Extracted pollen grains are foundadhered to the remains of the microsporangial walls(Fig. 6e-f) and are clearly boat-shaped and variouslyfolded. Individually extracted grains (Fig. 7) show arange of lengths and surfaces ornamentation. The grainsare 19–30 μm in maximum length (using SEM, x = 4).They are boat-shaped and ‘trisulcate’, i.e. monosulcatewith two pseudosulci present. The sulcus has a mem-brane that can be microrugulate to smooth and finelyperforate (Fig. 7a-d). The remaining surface is finelyperforate and can be smooth to almost microrugulate(Fig. 7e-g) between the perforations; sometimes there

are microfossulae (Fig. 7h-i). Based on the combinationof these features, the pollen closely resembles the dis-persed Eucommiidites-type pollen.

Fourier-transform infrared (FTIR) analysis of Cratoformation ambersThe FTIR spectra of both the extracted in situ amberfrom Araripestrobus gen. nov. and of the Brachyphyllumspecimen (Fig. 8) are typical for fossil resins. The im-portant insight is that the Araripestrobus gen. nov.spectrum is quite distinct from that of Brachyphyllum.The main features of the spectra are reported in Table 2.The broad absorption band with a maximum near

3400 cm− 1 in Araripestrobus gen. nov. is due to stretch-ing motions of O-H bonds, in part explained by atmos-pheric moisture absorbed by the sample during thepreparation for the analytical procedure, or as result ofoxidation by atmospheric oxygen [41]. In both spectra,the strong band at 2940 cm− 1 is due to the stretching ofaliphatic carbon-hydrogen bonds [2] and is considereddiagnostic of resinous structures [42]. Bending of thesame bonds produces the absorption peaks at around1450 cm− 1 and 1380 cm− 1 [2]. Another absorption bandis at around 1700 cm− 1, the so-called “carbonyl band”[2], also a feature typical of all fossil resins, caused bystretching of carbon-oxygen double bonds.The above described absorption bands are found in all

fossil resins, and therefore are not of particular diagnos-tic interest, but confirm the occurrence of a fossil resin.The upper part of the infrared spectrum, higher than1250 cm− 1, is difficult to interpret in terms of chemicalstructure [2], but it is more useful than the lower regionof the spectrum because it varies among different resins.In the FTIR spectra of the amber, the region between1250 and 1000 cm− 1 shows absorption bands caused bycarbon-oxygen single bonds [2, 43]. The vibrations ofthese bonds are influenced by the carbon skeleton of the

Fig. 8 FTIR spectra of extracted in situ Crato ambers from Araripestrobus resinosus gen. nov et sp. nov. and Brachyphyllum obesum. a Full FTIRspectra. b Detail of the fingerprint region of the spectra


whole molecule, and so it is difficult to assign the ab-sorption bands of this region to a specific structure.However, the region from 1250 to 625 cm− 1 is particu-larly useful as a “fingerprint” of any amber [2].An additional weak band at (1654 cm− 1), due to

stretching of double carbon-carbon bonds was foundin Araripestrobus gen. nov. but appears as a shoulderon the peak at 1690 cm− 1in the Brachyphyllumamber. Another very weak absorption band was

found at 878 cm− 1 in Araripestrobus gen. nov. andin Brachyphyllum ambers. This band, compatiblewith the 11.3 μm band described by [2, 44], is due toout-of-plane bending movements of two hydrogenatoms in a terminal methylene group, as a feature ofresin acids. This band is typically lacking or weak inolder resins, but can also be due to rapid oxidationof the methylene group after exposure to atmos-pheric oxygen [2, 44].

Table 2 Main absorption peaks (FTIR spectra) of extracted ambers from plant fossils from the Crato Formation

Functional group Band, wavelength [μm] Band, wavenumber [cm−1] Intensity Assignment

O-H 2.95 3390 medium Stretching of O-H bonds

-CH2 -CH3 3.40 2940 strong Stretching of C-H bonds

-CH2 -CH3 6.90 1450 medium Scissoring and bending of C-H bonds

-CH2 -CH3 7.25 1380 medium Bending of C-H bonds

-CH2 -CH3 11.39 878 weak C=CH2: out-of-plane bending of H interminal methylene group

-C=O 5.80 1724 medium Stretching of C=O double bonds

-C-O- 8.15, 8.60 1227, 1163 medium/weak Absorption of C-O single bonds

-C-O- 9.60, 9.85 1042, 1015 weak Absorption of C-O single bonds (alcohol)

C=C 6.05 1654 weak C=C stretching

-C-H aromatic 12.10, 14.10 826, 709 weak Out-of-plane bending of aromatic C-H

Fig. 9 Ion chromatograms of in situ Araripestrobus resinosus gen. nov et sp. nov. amber analyses with Pyr-GC-MS. a Total ion chromatogram from thesample. b Detail of the chromatogram with numbered peaks for identification. c Structures identified, numbers correspond to those numbered peaks in b



Pyrolysis gas chromatography mass spectroscopy of in situamber from Araripestrobus resinosus gen. nov. et sp. novThe amber is a mature class Ib amber, which has a regularpolylabdanoid structure lacking succinic acid, with minoramounts of various fatty acid products also present, butno palaeobotanically-useful biomarkers (Fig. 9).

DiscussionPalaeobotanical significance of Araripestrobus gen. novCone architectureBased on the conspicuous morphology of the slender wedge-shaped microsporophylls and their facetted arrangement (seeFigs. 1, 2a, 3c, 4), both the almost complete cone specimens(Fig. 2) and the detached cone portions (Figs. 3, 4 and 5) be-long to the same taxon without any doubt. The long axes ofthe detached cone portions (54–65mm) correspond in theirdimensions to the widths of the cones (maximum width 63–74mm). From the complete cones it is visible that each coneportion contains two to four horizontal levels of microsporo-phylls (see Fig. 1 for terminology); the same number of levelsis observed from the detached cone portions which displaythe cone in cross-sections.The different shapes of the basalmost and apicalmost

cone portions could putatively depend on a denser ar-rangement of not fully developed cone portions, but thecomplete cones are not well enough preserved for aconfident statement of this fact. Our conclusion, thatcones with their layered appearance of groups of micro-sporophylls and we find these layered parts as disc-shaped remains when preserved as transverse detachedcone portions, implies that either these disc-shapedportions are easily shed from the cone axis, or the coneincluding its axis disintegrated into these portions. Thefirst hypothesis is supported by the observations that (1)the paratype 2 displays the state of beginning disarticula-tion of cone portions and (2) that some cone portionslack axis tissue but show a hole instead. Although thecone peduncle superficially appears to be robust and thecone itself too, simply based on its size, the original tis-sue was obviously much softer than compared to fullywoody organs. The sigmoidal deformation of the coneportions, as seen in the complete cones (Fig. 2), and thelateral-longitudinal flattening of the complete cones in-dicate a non-woody or not completely woody nature ofthese microsporangiate organs. A single specimen(Fig. 2f) displays parts of the “naked” cone axis. Itclearly indicates a helical phyllotaxis of the microspo-rophylls but does not expose whether the cone axis issegmented or not, corresponding to the layered groupsof microsporophylls anticipating disintegration intocone portions. Calculated from the diagrammatic re-construction of a complete cone based on a compositeof specimens, a detached cone portions encompasses

about 120–160 microsporophylls, and a complete coneabout 1450–1850.Furthermore, we are convinced that these cones are

microsporangiate organs because they lack any ovule orseed but yield clusters of pollen. The detailed biology ofthese organs cannot be completely described becausethe original location where pollen was produced, thepollen sacs (microsporangia), cannot be clarified withthe studied specimens. If the detached cone portions areindeed shed from the axis they were obviously matureand pollen was likely at least partly already released.That could be the reason why not all detached cone por-tions yield in situ pollen in addition to being found inweathered sediments. Another possibility is that as themature cone degrades the microsporophylls start to‘clump together’ building distinct horizontal gaps andform these segmented portions as the cone decays. Gapscould be formed to better release pollen which couldalso explain why there are no pollen sacs left in thedetached cone portions. Invisibility of (free) pollen sacsin our cone material could be a hint that pollen sacswere embedded in microsporophyll tissue, similar toelongated embedded pollen sacs in Erdtmanithecaportulacensis [45]. Unfortunately, internal tissue of themicrosporophylls is not preserved in our studied de-tached cone portions.Based on cone architecture, with the cone appearing

to have layered groups of microsporophylls, and disinte-grating into distinctive detached portions of non-peltatemicrosporophylls, the new taxon does not resemble anyknown fossil taxon, neither described from the CratoFormation flora nor from other Cretaceous assemblages.Its characters are also clearly dissimilar from any pollen-producing structure in extant gymnosperms, of whichCycadales, Gnetales and Pinales are briefly discussedbelow. In particular, the cone architecture is ratherdissimilar to any known cone types from late Mesozoicconifers and gnetaleans as well as from microsporangiateorgans of early angiosperms. Only bennettitalean andcycadalean structures seem to have a superficially similararchitecture but are bisexual structure in case of bennet-titaleans. These pollen cones give evidence of a hithertounknown gymnospermous plant within the Crato For-mation, however, the affinities of the cones from solelytheir morphology was unclear. Previous studies of theCrato Formation flora had shown the presence of gneta-leans and conifers [46], and cycadophytes [47], the lattergroup is only known from the pollen record.Although the reproductive biology of the microsporo-

phylls present in Araripestrobus gen. nov. is notcompletely known, these simple reproductive units aredissimilar to equivalent organs of extant Gnetales. InEphedra, pollen cones are composed of bracts thatenclose stalked antherophores, each of which consists of


two fused microsporophylls bearing fused microsporan-gia [48]. The compound pollen cones of Gnetum exhibita whorled phyllotaxis with collar-like structures with nu-merous microsporophylls at nodes markedly separatedby internodes [48]. Cones of some species bear also non-functional ovules. The compound pollen cone ofWelwitschia is, by origin, a bisexual organ with the stam-inate “flowers” and non-functional ovules. Its grossmorphology is characterized by dense decussate arrange-ment of the staminate “flowers” consisting of fusedbracts and whorled microsporangiophores [48].Many gnetalean and gnetalean-like fossils have been de-

scribed from the Crato Formation flora, those describedwith reproductive structures have opposite-decussatescaled small, slender pollen strobili, e.g. Welwitschiostro-bus murili [31]. The two putative gnetaleans from theCrato Formation flora are clearly distinct from our cone.Cearania heterophylla also has small slender pollenstrobili [22], and Cariria orbiculiconiformis has a tripletreproductive structure including a pair of orbicular seedcones and a basket-like pollen strobilus in between thetwo seed cones [33]. It is clear that a gnetalean affinity forour cones can be ruled out solely based on the very differ-ent cone shape and structure, and that no gnetalean hasamber preserved in situ. The recovered in situ pollen alsohas no known gnetalean affinities.Cycadaleans and bennettitaleans are assumed to be

rare or even absent from the megafossil component ofthe Crato Formation flora. They are proven present fromthe pollen assemblage [19, 47, 49]. Possible ovulatecones are present, 8 cm long and about 6 cm in diam-eter, that are possibly referable to the bennettitaleanWilliamsonia [26] but this had not been evidentlyshown. Other plants with less clear, but still likelygymnospermous affinity have also been described fromthe Crato Formation flora such as Novaolindia dubia[32], which as yet has no microsporangiate organs de-scribed. The sole seed fern taxon is thought to have af-finity to the Caytoniales as it has multiovulate cupules,but microsporangiate organs are also not known so far[26]. In situ amber has not been reported from any ofthese plants listed above.Cone architecture of Araripestrobus gen. nov. resem-

bles that of extant cycadalean plants. Rather uniformly,they produce cylindrical cones with numerous appar-ently vertically but helically arranged wedged-shaped topeltate microsporophylls bearing numerous groups (sori)of small globose microsporangia [50]. At maturity, thecones open in a quite different way in that individualmicrosporophyll apophyses separate one from anotherto release pollen through the gaps. Cones are oftencurved, and finally they are shed as entire entities.Among the conifers described from the Crato Forma-

tion are the Araucariaceae (Araucaria and Brachyphyllum

[9, 27]), and the Cheirolepidiaceae [9, 28, 29], and othertaxa of uncertain affinity, such as Lindleycladus [27]. Fromthe latter taxon, reproductive structures are still unknown.The Classopollis pollen of Cheirolepidiaceae is very dis-tinctive, and pollen cones of this family are comparativelysmall exhibiting peltate microsporophylls [51]. However,such cones have not been reported from the Crato Forma-tion flora so far. In general, pollen cones of extant Arau-cariaceae are composed of numerous peltatemicrosporophylls bearing elongate free microsporangiaproducing distinctive pollen types while fossil araucarianpollen cones are often rather small with fewer microspo-rophylls [52]. A putative Agathis-like pollen cone withpreserved resin canals with in situ fossil resin ([8]; Pl 2,Figs. 1 and 2) and a putative female araucarian cone ([8];Pl 2, Figs. 3, 4 and 5) were figured from the Crato Forma-tion. What is striking when comparing our pollen conesto that of [8] is that all three have amber infilling the resincanals, but morphologically they are very distinct. Arari-pestrobus gen. nov., despite being preserved in a differentorientation, and more usually disintegrates into portionsof a larger cone structure, also has differing shaped micro-sporophylls that are far smaller having hexagonal outlinesof the distal ends and smooth outer surfaces. Pinaceae,Podocarpaceae and Cupressaceae are only reported fromthe pollen record [49]. Male cones of these conifers are farsmaller than our cones and they are composed of mark-edly fewer microsporophylls, which is why any affiliationof Araripestrobus gen. nov. to these families can be ruledout. Pollen cones of Pinaceae [53] and Cupressaceae [54]bear peltate microsporophylls with free subpendulusmicrosporangia (pollen sacs) attached to the laminateheads of microsporophylls. The catkin-like pollen cones ofPodocarpaceae are composed of peltate microsporophyllswith two free microsporangia. In short, establishment of anew genus and species is justified by the distinctiveness ofour material compared to pollen cone architecture ofother extinct and extant gymnospermous groups.

In situ pollen and taxonomic affinitiesFrom the holotype of the new taxon, we extracted in situpollen that is assignable to the Eucommiidites-type,placing it within the Erdtmanithecales, a group knownonly from isolated reproductive organs, but whose affin-ities with other seed plants remains unclear [40]. Thepresence of dispersed Eucommiidites pollen in the mud-stones of the Crato Formation, along with a diverse paly-noflora including representatives of araucarian andcheirolepidiaceous conifers, gnetaleans, cycads, bennet-tiataleans, angiosperms, horsetails, ferns and freshwateralgae was noted [19]. Unfortunately, the pollen sacs werenot recovered, so we lack information on their place-ment, number, and morphology.


Eucommiidites pollen has been reported in situ incones with peltate microsporophylls (Erdtmanithecatexensis from the Cretaceous of North America [55],Erdtmanitheca portucalensis from the Early Cretaceousof Portugal [56], Eucommiitheca hirsuta from the EarlyCretaceous of Portugal [40, 56], and Bayeritheca hughesiifrom the Cenomanian of Bohemia in Central Europe[57]), and within Erdtmanispermum seeds [40, 55]. Fromthis, Friis and Pedersen [40] established the Erdtma-nithecales, an extinct gymnosperm group that is recog-nised solely from reproductive parts to date. Our taxondoes not completely agree with the stalked peltatemicrosporangiate unit diagnosis, but the presence of thein situ Eucommiidites pollen indicates that the order isbroader than originally thought. These other pollencones assigned to the Erdtmanithecales are rather smallerand show a distinct phyllotaxis, opposite-decussate in thegenus Eucommiitheca and radial or whorled in the genusErdtmanitheca [40]. For Bayeritheca, the phyllotaxis is lessclear [57]. In Erdtmanitheca portucalensis, the muchsmaller microsporophylls radiate from a central axis,although their barrel-shape with slightly depressed peltatehead morphology is similar to our cones. Interestingly, themost complete specimen of this fossil species is a fragmentthat could superficially correspond to a hypotheticalfragment of our detached cone portions. Mendes et al. [45]calculate the number of microsporophylls of a completecone of Erdtmanitheca portucalensis as 100–150, whichwould correspond to the number of microsporophylls ofour cone portions. However, microsporophylls of Erdtma-nitheca portucalensis are less than 2mm long.Araripestrobus gen. nov. is also the first evidence of

members of the Erdtmanithecales in the megafossil com-ponent of the Crato Formation and from South America.Megafossil remains of this group, namely microsporangi-ate cones and isolated seeds, were hitherto reportedfrom the Early Cretaceous of North America andEurope. However, other plants parts and therefore planthabitus and size are, as yet, undiscovered. Recently, Friiset al. [10] hypothetically concluded, based on the sizes ofmesofossils (isolated seeds) from their proposed BEGgroup (Bennettitales, Erdtmanithecales, Gnetales), thatplants like Drewria potomacensis [58] could representErdtmanithecales. If so, the Crato plant Cariria orbiculo-coniformis, which shows some remarkable similarities toDrewria [33], may also belong to this group. However,the reproductive organs are distinct and by far smallerthan Araripestrobus gen. nov.Eucommiidites pollen is ‘trisulcate’ with a broad distal

sulcus and two narrower additional ‘sulci’ about 120°away from the sulcus [59], here termed pseudosulci. Inthe original descriptions, the terms colpus and pseudo-colpus have been used, but we update the terminologyto sulcus and pseudosulcus as this is correct when

dealing with gymnospermous pollen. A sulcus is anelongated aperture distally located, whereas a colpus isan equatorial elongated aperture and in some descrip-tions the main ‘colpus’ is described as distal. In our newspecies, the pollen has a sulcus membrane with clearlyrounded ends. In Araripestrobus resinosus gen. nov. etsp. nov. the pollen is finely perforate varying fromsmooth to almost microrugulate between the perforations,occasionally micro-fossulae are seen. The variation in thepollen surface and its size make it different to all otherEucommiidites pollen species described so far (see [60]).Palynological studies of the Araripe Basin recorded

dispersed pollen grains assigned to Eucommiiditestroedssonii and E. minor, plus three other unnamedEucommiidites species [61]. However, the samples arethought to lack firm stratigraphic control, so the preciselithostratigraphic ages are not known and may includematerial that is older or younger than the Crato Forma-tion flora [19]. However, the in situ pollen is most simi-lar in size to the recovered E. troedssonii, but differs itsornamentation, whereas E. minor pollen is smaller andlaevigate [62]. The in situ pollen is also much larger thanthe three unnamed Eucommiidites species described;additionally, of these unnamed species, two are psilateand the third is lightly scabrate but almost spherical inoutline [61].The in situ pollen recovered is most similar in length

to, although slightly larger than, in situ pollen of Erdt-manitheca portucalensis, but in width to in situ pollenfound inside Erdtmanitheca texensis. In situ pollen ofErdtmanitheca texensis measures 20–25 μm long and15–19 μm wide, and has lateral pseudosulci that areshorter than the main sulcus. The pollen surface ismainly psilate but with a few prominent granules and as-sociated pits [55]. In situ pollen of Erdtmanitheca portu-calensis measures 16.0–27.2 μm× 11.9–16.4 μm and hasa well-defined sulcus, with slightly expanded roundedends. Two slit-like lateral pseudosulci, longer than thesulcus, are present more or less in the equatorial plane.Margins of the apertures are irregular and the aperturemembrane is verrucate-granular. The pollen wall is psi-late, although puncta are seen in some specimens [45].The pollen inside Eucommiitheca hirsuta is elliptical inequatorial outline, about 15–20 μm long and 10–12 μmwide, typically pointed or truncate at the ends, withthree parallel and almost equal length sulci, and asmooth and faintly foveolate pollen wall [40]. Eucommii-dites pollen discovered inside Erdtmanispermum balti-cum seeds is distinctly foveolate and much smaller(22 μm by 15 μm, [55, 60]) than our larger and finelyperforate in situ pollen.Bayeritheca hughesii is a single elongate cone that has

in situ Eucommiidites pollen in synangia, not separatesporangia, unlike Erdtmanitheca and Eucommiitheca


[57]. The pollen has one normally straight sulcus thatdoes not reach the equator, commonly with roundedends. The two pseudosulci are usually curved, occasion-ally joined at one end to form a single ring furrow,unlike other in situ Eucommiidites pollen, including thatreported here. The distal half of the grains is more flat-tened than the proximal half, which is convex. Thepollen grains are psilate in light microscopy, and finelygranulate with protruding globular elements [0.1–(0.3)0.4 μm] under SEM. The pollen grains are 14–16 (15.2)μm long; 10–13 (11.2) μm wide, smaller than the pollenreported here. Interestingly, the macerated microsporan-giate units have small resin bodies (40–65 μm indiameter) present [57]. Bayeritheca hughesii is, so far,the largest male reproductive structure described fromthe Erdtmanithecales but even these cones are conspicu-ously smaller than Araripestrobus gen. nov.Pollen grains resembling Eucommiidites were also found

in situ in cones of Hastyostrobus muirii from the MiddleJurassic of Yorkshire, England [63]. Based on the wallultrastructure, Tekleva et al. [64] believe that the pollen isassignable to Eucommiidites, but this was doubted byMendes et al. [45]. Resinous microsporophylls of the earlyCretaceous Loricanthus resinifer, initially appear to resem-ble one of our specimens (Fig. 2f), but they are peltate,borne in smaller strobili and have a more rounded (notEucommiidites–type) pollen grain in situ, so the affinity ofthe Loricanthus plant is unknown [65, 66].Our in situ pollen cannot be assigned to any currently

described Eucommiidites species. We refrain from givingour in situ pollen a name as the cones themselves arenamed. It is interesting to note that although we havepollen assignable to Eucommiidites, the reproductive conestructure is tremendously different from all other struc-tures bearing Eucommiidites-type pollen, in the layeredappearance and helical arrangement of the non-peltatemicrosporophylls. Additionally, the new taxon exhibitsprominent resin canals, a feature not described for othermembers of the Erdtmanithecales. This unusual feature,along with the differences in cone morphology may eitherindicate that the poorly known Erdtmanithecales encom-passes more diversity than originally thought, or that morethan one plant group produced Eucommiidites-typepollen. If the habitus of Erdtmanithecales is putativelyshrub, sub-shrub or herbaceous-like is assumed by [10]the latter might be the case because the large Araripestro-bus gen. nov. cones likely hint towards larger plants.

Amber classificationFTIR spectra of ambersAmber from Araripestrobus gen. nov. is similar to theBrachyphyllum amber measured here in parts of its gen-eral spectrum, but a key difference is the lack of a strongpeak at 1690 cm− 1, and the presence of a peak at 1645

cm− 1, which appears as a shoulder to the large peak at1690 cm− 1 present in Brachyphyllum. This suggests thatthere is a slight difference in the amber chemistry, but itis not clear if this has resulted from differences in theoriginal resin chemistries between the plants, or differ-ences in maturation or weathering. Maturational differ-ences cannot be excluded since all the plants are fromthe C6 limestone of the Crato Formation, but perhapsfrom different horizons that are distinguished by colour,grain size and other macroscopic rock features. Recentpublications on the Crato Formation reinforce these dif-ferences are likely caused by fluctuations in water depthand water chemistry [67–69]. Weathering differences area possibility too since different quarries at differentlocations in the Araripe Basin operate to retrieve thelimestones, and different specimens may have been alsoexposed after collection for different durations. Theprocess of resin maturation [70], also named amberiza-tion, causes changes in the chemical composition of fos-sil resins, and the rate of this phenomenon depends onseveral variables, such as temperature, age and thermalhistory of the sample. Resins with similar palaeobotan-ical origin may present different compositions, as indi-cated by infrared spectroscopy, in consequence ofseveral taphonomic variables. These results from FTIRspectrum can be further confirmed by Pyr-GC-MS ana-lysis which can give a more specific assignment of chem-ical structures of the fossil resin (see below).The in situ Crato Formation amber spectrum, with a bi-

phasic weak absorption band between 1240 cm− 1 and 1015cm− 1 is also partially similar to that of New Zealand amber(thought to originate from the araucarian genus Agathis,see [71]), and of extant kauri (Agathis australis) resin [2,71–73]. Conversely, the fingerprint region appears differentfrom that of present-day Pinus resin [72, 73].Further comparisons can be done with spectra from

other fossil resins, considering the fingerprint region,where absorption caused by carbon-oxygen single bonds,as well as aromatic ethers and phenols occurs. This re-gion from the in situ Crato Formation amber spectrumshows a pattern similar to that of Cedar Lake (Manitoba,Canada) Cretaceous amber and also to that of AtlanticCoastal Plain fossil resin ([2]; page 89 and plates XIV,XVI), attributed to family of Araucariaceae. However, re-cent FTIR analyses of the Grassy Lake ambers, alongwith fossil plant fragments found within the ambers,showed a closer relationship to Cupressaceae [74]. TheAtlantic Coastal Plain or Raritan (New Jersey) amberoriginated from the Cupressaceae [75], with additionalchemical evidence provided using Pyr-GC-MS [76].Our amber spectra appear to have strong affinities with

those of other Cretaceous presumed araucariacean-derived ambers, including Cretaceous ambers from Spain[77], and Cretaceous ambers from France [78]. Infrared


(IR) analyses of amber have been previously interpreted togive differing results in some cases; however, Tappertet al. [73], using micro-FTIR, showed that conifer resinscan effectively be classified into two groups: ‘pinaceousresins’ from Pinaceae, which consist mainly of abietane/pimarane diterpenes, and ‘cupressaceous resins’, whichthey associate with Cupressaceae, Sciadopityaceae, Arau-cariaceae, and Podocarpaceae, and consist mainly of lab-danoid diterpenes. Our ambers would fall in to‘cupressaceous resins’ group identified by Tappert et al.[73], which may have an araucariacean or cupressacean,but not a pinaceous affinity. Notably, no ambers fromother gymnosperm groups were known until now, so thisgrouping may have to be expanded to include fossil resinsfrom these enigmatic erdtmanithecalean plants.

Pyrolysis gas chromatography mass spectroscopyThe in situ amber belongs to the class Ib group of resins,which is the most common form of amber in thegeosphere. Class Ib ambers are formed by copolymers ofregular labdanoid diterpenes, especially communic acid,communol and biformenes. In this case, the distributionof products observed indicates that the macromolecularstructure of the resin is dominated by communic acid(products from communal and biformene are essentiallyabsent). No occluded intact diterpenes are observed,therefore, an affinity to the Pinaceae can be excluded astheir resins are rich mostly in abietane and pimaranediterpenic acids and lack significant amounts of free ormacromolecular labdanoids (see also [73, 79]). Wecannot exclude an affinity with the Araucariaceae orCupressaceae, which are rich in labdanes, includingcommunic acid and agathic acid [76, 80], nor the Podo-carpaceae, and Taxodiaceae. There are minor amountsof fatty acids, but no biomarkers present meaning that acloser identification of the source plant is not possible.The absence of intact occluded diterpenes and the highratio of C15/C14 bicylic products observed in the pyroly-sate derived from this amber suggests that it has experi-enced a moderate degree of thermal stress during itshistory.The assignment of the amber to class Ib excludes an

affinity with the highly resinous angiosperm Hymenaea,which forms class Ic ambers, which are linked to therelatively common, but much younger Dominican andMexican ambers. Class Ia ambers are Paleogene Balticambers which have succinic acid present [80], which isclearly absent from the amber analysed here. What isinteresting is that the amber has as similarity with aMesozoic amber found in archaeological context inSouth America [81], but for which the palaeobotanicalaffinities are unknown.Taken together, the FTIR analysis and the Pyr-GC-MC

indicate that there is no clear way to pinpoint to which

botanical group the resin from Araripestrobus gen. nov.can be chemically placed, only which groups (Pinaceae,Hymenaea) can be excluded. The presence of the in situpollen showing an erdtmanithecalean affinity, suggeststhat other gymnosperm source plants of amber have notbeen detected until now.

ConclusionsWe used multiple lines of evidence to uncover the bo-tanical affinity of cones and detached cone portions within situ amber and in situ pollen, and to understand thepotential amber sources of the Crato Formation flora,beyond the araucariacean source plants already known.The in situ pollen shows that Araripestrobus resinosusgen. nov. et sp. nov. is a microsporangiate organ ofgymnospermous affinity, displaying a very dissimilarmacromorphology to any pollen cones of extinct and ex-tant taxa described to date. Based on the presence of insitu Eucommiidites-type pollen, the new genus and spe-cies is placed in the order Erdtmanithecales, but basedon differences in the cone morphologies they cannot beplaced within the only hitherto known family, Erdtma-nithecaceae. Besides dispersed pollen, these specimensrepresent the first macrofossil evidence of the Erdtma-nithecales in South America. The FTIR analyses of thein situ amber implies an affinity with some conifers (ara-ucariacean or cupressacean), by effectively ruling out anaffinity with the Pinaceae. The Pyr-GC-MS analysisshows this amber to be a class Ib resin that has under-gone some thermal maturation. From this, it is clear thatthe new erdtmanithecalean taxon produced a resin thathas matured to an amber very similar to the labdane-based resins and ambers thought to derive from someconifer groups, particularly araucariaceans, cupressa-ceans, and cheirolepidiaceous conifers, but clearly ex-cludes a pinaceous affinity.

AbbreviationsBEG group: Bennettitales-Erdtmanithecales-Gnetales group; FTIR: Fourier-transform infrared; IR: Infrared; LM: Light microscopy; Pyr-GC-MS: Pyrolysis gaschromatography mass spectroscopy; SEM: Scanning electron microscopy

AcknowledgementsWe gratefully acknowledge the help and guidance from Reinhard Zetter(Vienna) in the single pollen grain preparation, pollen description andscanning electron microscopy. We warmly thank Christa Hofmann (Vienna)for help with the pollen description and scanning electron microscopy. Wethank Dorothea Hause-Reitner (Göttingen) for assistance with field emissionscanning electron microscopy. Thanks also to Giovanni Marzaro (Padova) forhelp with FTIR analysis. Márcio Mendes (Fortaleza) kindly provided photo-graphs of the specimen from the Phoenix Foundation. Barbara Mohr (Berlin)is kindly thanked for her ideas and allowing access to specimens. VolkerWilde (Frankfurt) kindly gave access to collection specimens as well and dis-cussed general palaeobotanical details with us. Furthermore, we thank JiříKvaček (Prague) for sharing knowledge on erdtmanithecalean plants with us.We would also like to thank the editor and both anonymous reviewers fortheir supportive comments that strengthened the manuscript. We gratefullyacknowledge that open access was funded by the Open Access PublishingFund of the University of Vienna.


Authors’ contributionsLJS, EAR, ARS and LK conceived the research. LK, ARS, LJS and EAR preparedthe specimens. KBA, EAR and ER performed analyses on the extracted amber.LJS and EAR extracted and analysed the pollen and co-wrote the draftmanuscript. DRN and WFS reviewed the geology of the host rocks. Allauthors made significant inputs, commented on and approved the finalversion of this manuscript.

FundingContribution by LJS funded in part by the DeutscheForschungsgemeinschaft (DFG grant SE 2335/3). Contributions by LK, DRN,WFS are partly supported by the CAPES-DAAD PROBRAL mobility program(CAPES ID 88881.198776/2018–01; DAAD ID-57446885). The funding bodieshad no role in the design of the study nor collection, analysis, andinterpretation of data and the writing of the manuscript.

Availability of data and materialsAll specimens are available from their listed repositories: Museum fürNaturkunde Berlin, University of Portsmouth Collection, SenckenbergResearch Institute and Natural History Museum Frankfurt/M., UniversidadeEstado de Rio de Janeiro, and Fundação Paleontológica Phoenix, Ceará,Brazil.

Ethics approval and consent to participateNot applicable as all specimens present in public repositories.

Consent for publicationNot applicable.

Competing interestsThere are no competing interests.

Author details1Department of Palaeontology, University of Vienna, Althanstraße 14, 1090Vienna, Austria. 2School of the Environment, Geography and Geosciences,University of Portsmouth, Burnaby Road, Portsmouth PO1 3QL, UK.3Department of Geobiology, University of Göttingen, Goldschmidtstraße 3,37077 Göttingen, Germany. 4Department of Pharmaceutical andPharmacological Sciences, University of Padova, Largo E. Meneghetti 2,35131 Padova, Italy. 5School of Earth Systems and Sustainability, SouthernIllinois University, Carbondale, IL 62901, USA. 6Department of Geology,Federal University of Ceará, Fortaleza, CE 60440-554, Brazil. 7SenckenbergNatural History Collections Dresden, Museum of Mineralogy and Geology,Koenigsbruecker Landstraße 159, 01109 Dresden, Germany.

Received: 14 February 2020 Accepted: 6 July 2020

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